Deposition of laminin and fibronectin in dysraphic mutant mice: an immunofluorescent study

Abnormal loop-tail (Lp/Lp) mutant mouse embryos exhibiting severe exencephaly and myeloschisis were analyzed and compared with their normal (+/+; Lp/+) littermates by means of immunofluorescence histochemistry to determine regional differences in the distribution of laminin (L) and fibronectin (FN). In the neural basement membrane and adjacent mesenchymal cell matrix of the abnormal embryos, regional differences in the deposition of L and FN were similar to those in normal littermates. Moreover, most of the putative neural crest (NC) cells appeared to emigrate normally in terms of their site of detachment and migration pathways, despite the severe topographic distortions and loss of neuroepithelial integrity. However, some putative NC cells projected incorrectly from the 'luminal' surface of the neuroepithelium, suggesting that some of the NC may be abnormal or sequestered and prevented from appropriate detachment and emigration from the neural tube.     

Distribution of laminin in the developing peripheral nervous system of the chick

During axonal elongation in the developing peripheral nervous system, the temporal and spatial distribution of adhesive molecules in extracellular matrices and on neighboring cell surfaces may provide "choices" of pathways for growth cone migration. The extracellular matrix glycoprotein laminin appears in early embryos and mediates neuronal adhesion and neurite extension in vitro. In this study, we have examined the distribution of laminin at early periods of peripheral nervous system development. The distribution of laminin, demonstrated by immunostaining frozen sections of chick embryos, was compared to the distribution of fibronectin and of early peripheral neurites as revealed with an antibody to a neurofilament-associated protein. Laminin is present in the neural tube basement membrane, in early ganglia, and in developing dorsal and ventral roots, where the laminin staining pattern parallels that of neurofilaments. In early ganglia and nerve roots, laminin immunostaining defines loose "meshworks" rather than basement membranes, which seem to form slightly later in these structures. In contrast, fibronectin is absent in neural tube basement membrane, ganglia, and nerve roots, although it is present along neural crest migratory pathways and in intersomitic spaces. Our observations of laminin distribution are consistent with the possibility that laminin provides an adhesive surface for neurite extension at some stages of early peripheral nervous system development.     

Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen

The trunk neural crest originates by transformation of dorsal neuroepithelial cells into mesenchymal cells that migrate into embryonic interstices. Fibronectin (FN) is thought to be essential for the process, although other extracellular matrix (ECM) molecules are potentially important. We have examined the ability of three dimensional (3D) ECM to promote crest formation in vitro. Neural tubes from stage 12 chick embryos were suspended within gelling solutions of either basement membrane (BM) components or rat tail collagen, and the extent of crest outgrowth was measured after 22 hr. Fetal calf serum inhibits outgrowth in both gels and was not used unless specified. Neither BM gel nor collagen gel contains fibronectin. Extensive crest migration occurs into the BM gel, whereas outgrowth is less in rat tail collagen. Addition of fibronectin or embryo extract (EE), which is rich in fibronectin, does not increase the extent of neural crest outgrowth in BM, which is already maximal, but does stimulate migration into collagen gel. Removal of FN from EE with gelatin-Sepharose does not remove the ability of EE to stimulate migration. Endogenous FN is localized by immunofluorescence to the basal surface of cultured neural tubes, but is not seen in the proximity of migrating neural crest cells. Addition of the FN cell-binding hexapeptide GRGDSP does not affect migration into either the BM gel or the collagen gel with EE, although it does block spreading on FN-coated plastic. Thus, although crest cells appear to use exogenous fibronectin to migrate on planar substrata in vitro, they can interact with 3D collagenous matrices in the absence of exogenous or endogenous fibronectin. In BM gels, the laminin cell-binding peptide, YIGSR, completely inhibits migration of crest away from the neural tube, suggesting that laminin is the migratory substratum. Indeed, laminin as well as collagen and fibronectin is present in the embryonic ECM. Thus, it is possible that ECM molecules in addition to or instead of fibronectin may serve as migratory substrata for neural crest in vivo.     

Distribution of fibronectin in the early phase of avian cephalic neural crest cell migration

Immunofluorescence and immunoperoxidase labeling for fibronectin was used to study the early events of cephalic neural crest cell migration in avian embryos. Prior to crest cell appearance, fibronectin was associated with the basement membranes of all tissues. The loose mesenchymal cells were also surrounded by this glycoprotein. The crest cell individualization phase included a transient rounding up and a rapid increase in cell number in a very limited space. Whereas the neural tube basement membrane was not formed dorsally at the site of emergence of crest cells, it was partially fused laterally with the ectoderm basement membrane apparently preventing immediate crest cell emigration. Further increase in cell number occurred concomitantly with their penetration between the two developing basement membranes of the neural tube and the ectoderm. The localization of migrating crest cells is apparently greatly influenced by local interactions between the ectoderm and the neural tube, whose morphogenesis differs considerably at each axial level: at the mesencephalic-rhombencephalic levels, crest cells rapidly reached a cell-free space that was mostly devoid of fibronectin. Further migration occurred laterally in that space while pioneer crest cells became surrounded by fibronectin in their environment. Crest cells progressed as a confluent multicellular layer with an apparent velocity of 70 μm/hr. At the prosencephalic and median rhombencephalic levels, crest cells accumulated between the fibronectin-rich basement membranes of the ectoderm and the neural tube. Pioneer crest cells were arrested at the site of attachment of the ectoderm and the neural tube basement membranes (i.e., optic vesicles and otic placodes). Crest cells resumed their migration when more space became available during the constriction of the optic vesicles and the invagination of the otic placodes.     

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.     

Collagen I, laminin, and tenascin: ultrastructure and correlation with avian neural crest formation.

We have investigated the distribution of type I collagen, tenascin, and laminin in younger chick embryos than have previously been studied in detail. The initial appearance of type I collagen, but not tenascin and laminin, is exactly correlated with the beginning of neural crest migration, suggesting a role for collagen I in the migration. Light microscopy of whole mounts of 2-day-old chick embryos reveals that type I collagen is expressed in a rostral to caudal gradient; it localizes to the notochord sheath before accumulating around the neural tube and somites. Collagen I and tenascin also associate with central somite cells. Surprisingly, no extracellular matrix can be detected among the early sclerotomal cells, which suggests that little or no cell migration is involved in this epithelial-mesenchymal transformation. Electron microscopy using peroxidase antiperoxidase reveals that tenascin is present in nonstriated, 10 nm wide fibrils and in interstitial bodies, both of which have previously been reported to contain fibronectin. However, collagen I only occurs in the 10 nm fibrils and larger striated fibrils. This is the first ultrastructural study to assign tenascin to fibrils and interstitial bodies and to describe its appearance and disappearance from embryonic basement membranes. The discussion emphasizes the possible importance of type I collagen in neural crest cell migration and compares the ultrastructural associations of the ECM molecules present at this early embryonic stage.     

Aberrant convergence of the neural folds in the mouse mutant vl.

Progressive changes in the dorsolateral angles (DA) and ventral angle (VA) during elevation and convergence of the caudal neural folds were morphometrically analyzed in normal and dysraphic abnormal embryos of the mouse mutant vacuolated lens (vl), and correlations with the configuration of microfilaments in the apices of neuroepithelial cells were made by means of ultrastructural cytochemistry. In 22-28 somite stage abnormal (vl/vl) embryos, the DA and VA are larger than those in their normal counterparts at each comparable level of the caudal neural folds, suggesting that defective convergence involves both the DA and VA in this mutant. In 30-35 somite stage abnormal embryos, the VA is likewise larger than that in normal embryos in which the neural folds have converged and closed; however, the DAs are much smaller, indicating that a medial collapse of the dorsal ends of the neural folds may occur secondary to the closure failure. At the DA, the ultrastructural configuration of microfilaments is similar in abnormal and normal embryos in terms of their circumferential arrangement around the perimeters of the neuroepithelial cell apices. In abnormal embryos, however, the bundles of microfilaments are more delicate and less prominent than in normal embryos; thus it is possible that a quantitative and/or functional deficiency in these elements may be involved in the failure of the abnormal neuroepithelium to bend properly during convergence of the neural folds.     

Fibronectin in early avian embryos: Synthesis and distribution along the migration pathways of neural crest cells

Summary Immunoperoxidase labelling for fibronectin (FN) in chick embryos showed FN-positive basement membranes surrounding the neural crest cell population prior to crest-cell migration. At cranial levels, crest cells migrated laterally into a large cell-free space. Initially they moved as a tongue of cells contacting the FN-positive basement membrane of the ectoderm, but later the crest cell population expanded into space further from the ectoderm, until eventually the entire cranial cell-free space was occupied by mesenchyme cells. This was accompanied by the appearance of FN among the crest cells. At trunk levels, crest cells entered a relatively small space already containing FN-positive extracellular material. At later stages the migration of trunk crest cells broadly matched the distribution of FN. In vitro, chick and quail embryo ectoderm, endoderm, somites, notochord and neural tube synthesized and organized fibrous FN-matrices, as shown by immunofluorescence. Ectoderm and endoderm deposited this matrix only on the substrate face. The FN content of endoderm and neural tube matrices was transient, the immunofluorescence intensity declining after 1–2 days in culture. Some crest cells of cranial and sacral axial levels synthesized FN. Our data suggests that these were the earliest crest cells to migrate from these levels. This ability may be the first expression of mesenchymal differentiation in these crest cells, and in vivo enable them to occupy a large space. Almost all crest cells from cervico-lumbar axial levels were unable to synthesize FN. In vivo, this inability may magnify the response of these crest cells to FN provided by the neighbouring embryonic tissues.     

Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo

Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina.     

Avian vasculogenesis and the distribution of collagens I, IV, laminin, and fibronectin in the heart primordia

The heart-forming regions of the early embryo are composed of splanchnic mesoderm, endoderm, and the associated ECM. The ECM of the heart-forming regions in stage 7-9 chicken embryos was examined using immunofluorescence. Affinity purified antibodies to chicken collagens type I and IV, chicken fibronectin, and mouse laminin were used as probes. We report that (1) the basement membrane of the endoderm contains immunoreactive laminin and collagen IV; (2) the nascent basement membrane of the heart splanchnic mesoderm contains immunoreactive laminin, but not type IV collagen, and (3) the prominent ECM between the splanchnic mesoderm and the endoderm (the primitive-heart ECM) contains collagen IV, collagen I, fibronectin, but not laminin. In addition, we describe microscopic observations on the spatial relationship of cardiogenic cells to the primitive-heart ECM and the endodermal basement membrane.     

Perturbation of cranial neural crest migration by the HNK-1 antibody

The HNK-1 antibody recognizes a carbohydrate moiety that is shared by a family of cell adhesion molecules and is also present on the surface of migrating neural crest cells. Here, the effects of the HNK-1 antibody on neural crest cells were examined in vitro and in vivo. When the HNK-1 antibody was added to neural tube explants in tissue culture, neural crest cells detached from laminin substrates but were unaffected on fibronectin substrates. In order to examine the effects of the HNK-1 antibody in vivo, antibody was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. The injected antibody persisted for approximately 16 hr on the injected side of the embryo and appeared to be most prevalent on the surface of neural crest cells. Embryos fixed within the first 24 hr after injection of HNK-1 antibodies (either whole IgMs or small IgM fragments) showed one or more of the following abnormalities: (1) ectopic neural crest cells external to the neural tube, (2) an accumulation of neural crest cell volume on the lumen of the neural tube, (3) some neural tube anomalies, or (4) a reduction in the neural crest cell volume on the injected side. The ectopic cells and neural tube anomalies persisted in embryos fixed 2 days postinjection. Only embryos having 10 or less somites at the time of injection were affected, suggesting a limited period of sensitivity to the HNK-1 antibody. Control embryos injected with a nonspecific antibody or with a nonblocking antibody against the neural cell adhesion molecule (N-CAM) were unaffected. Previous experiments from this laboratory have demonstrated than an antibody against integrin, a fibronectin and laminin receptor caused defects qualitatively similar to those resulting from HNK-1 antibody injection (M. Bronner-Fraser, J. Cell Biol., 101, 610, 1985). Coinjection of the HNK-1 and integrin antibodies resulted in a greater percentage of affected embryos than with either antibody alone. The additive nature of the effects of the two antibodies suggests that they act at different sites. These results demonstrate that the HNK-1 antibody causes abnormalities in cranial neural crest migration, perhaps by perturbing interactions between neural crest cells and laminin substrates.     

Distribution of integrins and their ligands in the trunk of Xenopus laevis during neural crest cell migration

We have examined the distribution in Xenopus embryos of beta 1 subunits of integrin, as recognized by cross-reactive antibodies against the avian integrin beta 1 subunit. These antibodies recognize a doublet of bands of approximately 120 kD in Xenopus embryos. The distribution pattern of these integrin cell surface receptors was compared with that of two possible ligands, fibronectin and laminin, in the extracellular matrix during the time of neural crest cell migration. Integrin immunoreactivity in the early neurula was observed lightly outlining somite and epidermal cells and the notochord. The integrin immunostaining increased with developmental age and was observed on most cell types in the embryo but was particularly notable in the intersomitic clefts through which motoraxons grow. The immunoreactivity in this region was not, however, wholly on the axon surfaces, since intersomitic integrin remained detectable in embryos in which the neural tube had been ablated. Fibronectin and laminin were more extensively distributed than integrin at all stages examined. Immunoreactivity for both was observed around the neural tube, notochord, somites, epidermis, dorsal mesentery, and lateral plate mesoderm. The distribution of laminin and fibronectin around the somites was particularly interesting since it was non-uniform and similar to that of integrin. Strongest staining was observed in the intersomitic clefts, and weakest staining was observed on the medial surface of the somites, which faces the neural tube and notochord. The major differences in distribution pattern between the fibronectin and laminin immunoreactivities were that only fibronectin was detected in the mesenchyme of the dorsal fin. Our results demonstrate that a molecule homologous to avian integrin is present in Xenopus embryos during neural crest cell migration and motoraxon outgrowth. Its presence in the intersomitic clefts and on the surface of many embryonic cell types together with the abundant distribution of its ligands are consistent with a potentially important developmental function in neurite outgrowth and/or muscle development.     

The neural crest in presomite to 40-somite murine embryos

The production of cells by the neural crest is studied light-microscopically in 10 microns and 1 micron serially sectioned mouse and rat embryos, ranging in age from presomite to 40-somite stages. In the head region, mesectoderm formation starts in a pre-neural plate stage. It continues to the 20-somite stage. This implies that the contribution of the neural crest to the head mesoderm must be considerable. In the trunk, the neural crest only produces cells after adhesion of the neural walls. Mesectoderm formation continues for a long time, slowly retreating in a caudal direction. At the 40-somite stage, mesectoderm formation still occurs in the most caudal part of the trunk. Compared to the head, the contribution of the neural crest in the trunk seems to be less important than that of the primitive streak.     

An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt

Fibronectin and laminin are two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation describes changes in their distribution during regeneration of the newt forelimb, as determined by indirect immunofluorescence. The distribution of fibronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myofibers corresponding to its basement membrane; the perineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithelium, and dermal glands. The cytoplasm of myofibers, axons, skin epithelium, and bone matrix lacked fluorescence for both glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, blastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the myoblast fusion zone. Laminin was first observed around the regenerated myotubes; this was followed by the appearance of fibronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distribution of laminin and fibronectin was similar to that seen in normal limb. Based on the distribution pattern of these glycoproteins, it is concluded that fibronectin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fibronectin may be important in the determination of the architecture of the regenerated limb.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

An antibody to a receptor for fibronectin and laminin perturbs cranial neural crest development in vivo

Previous studies from this laboratory (M. Bronner-Fraser (1985). J. Cell Biol. 101, 610) have demonstrated that an antibody to a cell surface receptor complex caused alterations in avian neural crest cell migration. Here, these observations are extended to examine the distribution and persistency of injected antibody, the dose dependency of the effect, and the long-term influences of antibody injection. The CSAT antibody, which recognizes a cell surface receptor for fibronectin and laminin, was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. Injected antibody molecules did not cross the midline, but appeared to diffuse throughout the injected half of the mesencephalon, where they remained detectable by immunocytochemistry for about 22 hr. Embryos were examined either during neural crest migration (up to 24 hr after injection) or after formation of neural crest-derived structures (36-48 hr after injection). In those embryo fixed within the first 24 hr, the major defects were a reduction in the neural crest cell number on the injected side, a buildup of neural crest cells within the lumen of the neural tube, and ectopically localized neural crest cells. In embryos allowed to survive for 36 to 48 hr after injection, the neural crest derivatives appeared normal on both the injected and control side, suggesting that the embryos compensated for the reduction in neural crest cell number on the injected side. However, the embryos often had severely deformed neural tubes and ectopic aggregates of neural crest cells. In contrast, several control antibodies had no effect. These findings suggest that the CSAT receptor complex is important in the normal development of the neural crest and neural tube.     

Distribution of laminin and collagens during avian neural crest development

The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscle. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues. The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and lamin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development. These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny. (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration. (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis. (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.     

A monoclonal antibody against a laminin-heparan sulfate proteoglycan complex perturbs cranial neural crest migration in vivo

INO (inhibitor of neurite outgrowth) is a monoclonal antibody that blocks axon outgrowth, presumably by functionally blocking a laminin-heparan sulfate proteoglycan complex (Chiu, A. Y., W. D. Matthew, and P. H. Patterson. 1986. J. Cell Biol. 103: 1382-1398). Here the effect of this antibody on avian neural crest cells was examined by microinjecting INO onto the pathways of cranial neural crest migration. After injection lateral to the mesencephalic neural tube, the antibody had a primarily unilateral distribution. INO binding was observed in the basal laminae surrounding the neural tube, ectoderm, and endoderm, as well as within the cranial mesenchyme on the injected side of the embryo. This staining pattern was indistinguishable from those observed with antibodies against laminin or heparan sulfate proteoglycan. The injected antibody remained detectable for 18 h after injection, with the intensity of immuno-reactivity decreasing with time. Embryos ranging from the neural fold stage to the 9-somite stage were injected with INO and subsequently allowed to survive for up to 1 d after injection. These embryos demonstrated severe abnormalities in cranial neural crest migration. The predominant defects were ectopic neural crest cells external to the neural tube, neural crest cells within the lumen of the neural tube, and neural tube deformities. In contrast, embryos injected with antibodies against laminin or heparan sulfate proteoglycan were unaffected. When embryos with ten or more somites were injected with INO, no effects were noted, suggesting that embryos are sensitive for only a limited time during their development. Immunoprecipitation of the INO antigen from 2-d chicken embryos revealed a 200-kD band characteristic of laminin and two broad smears between 180 and 85 kD, which were resolved into several bands at lower molecular mass after heparinase digestion. These results indicate that INO precipitates both laminin and proteoglycans bearing heparan sulfate residues. Thus, microinjection of INO causes functional blockage of a laminin-heparan sulfate proteoglycan complex, resulting in abnormal cranial neural crest migration. This is the first evidence that a laminin-heparan sulfate proteoglycan complex is involved in aspects of neural crest migration in vivo.     

Demonstration of laminin, a basement membrane glycoprotein, in routinely processed formalin-fixed human tissues

Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 67:73-78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.     

Regional differences in the expression of laminin isoforms during mouse neural tube development

Many significant human birth defects originate around the time of neural tube closure or early during post-closure nervous system development. For example, failure of the neural tube to close generates anencephaly and spina bifida, faulty cell cycle progression is implicated in primary microcephaly, while defective migration of neuroblasts can lead to neuronal migration disorders such as lissencephaly. At the stage of neural tube closure, basement membranes are becoming organised around the neuroepithelium, and beneath the adjacent non-neural surface ectoderm. While there is circumstantial evidence to implicate basement membrane dynamics in neural tube and surface ectodermal development, we have an incomplete understanding of the molecular composition of basement membranes at this stage. In the present study, we examined the developing basement membranes of the mouse embryo at mid-gestation (embryonic day 9.5), with particular reference to laminin composition. We performed in situ hybridization to detect the mRNAs of all eleven individual laminin chains, and immunohistochemistry to identify which laminin chains are present in the basement membranes. From this information, we inferred the likely laminin variants and their tissues of origin: that is, whether a given basement membrane laminin is contributed by epithelium, mesenchyme, or both. Our findings reveal major differences in basement composition along the body axis, with the rostral neural tube (at mandibular arch and heart levels) exhibiting many distinct laminin variants, while the lumbar level where the neural tube is just closing shows a much simpler laminin profile. Moreover, there appears to be a marked difference in the extent to which the mesenchyme contributes laminin variants to the basement membrane, with potential contribution of several laminins rostrally, but no contribution caudally. This information paves the way towards a mechanistic analysis of basement membrane laminin function during early neural tube development in mammals.